CN117500828A

CN117500828A - Bispecific binding molecules that bind VEGF and Ang2 and uses thereof

Info

Publication number: CN117500828A
Application number: CN202280039743.2A
Authority: CN
Inventors: 黎一鸣; 胡思怡; 荆华
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Innovent Biologics Suzhou Co Ltd
Priority date: 2021-06-04
Filing date: 2022-06-02
Publication date: 2024-02-02
Also published as: CA3221192A1; EP4349861A1; KR20240023095A; WO2022253314A1; TW202302633A; IL309035A; AU2022287100A1

Abstract

Antibodies directed against vascular endothelial growth factor (VEGF/VEGF-A) and against angiopoietin-2 (ANG-2), respectively, as well as bispecific binding molecules (e.g., antibodies) directed against vascular endothelial growth factor (VEGF/VEGF-A) and angiopoietin-2 (ANG-2), respectively, as well as methods of making the same, pharmaceutical compositions comprising the antibodies or molecules, and uses thereof.

Description

Bispecific binding molecules that bind VEGF and Ang2 and uses thereof

The present invention relates to antibodies directed against vascular endothelial growth factor (VEGF/VEGF-A) and against angiopoietin-2 (ANG-2), respectively, and bispecific binding molecules (e.g. antibodies) directed against vascular endothelial growth factor (VEGF/VEGF-A) and angiopoietin-2 (ANG-2), respectively, as well as methods for their preparation, pharmaceutical compositions comprising said antibodies or molecules, and uses thereof.

Background

Angiogenesis is involved in the pathogenesis of a variety of diseases including solid tumors, diseases associated with intraocular neovascularization, rheumatoid arthritis (rheumatoid arthritis), and psoriasis.

VEGF is a potent and ubiquitous vascular growth factor. VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) and endocrine gland derived VEGF (EG-VEGF). The active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members. VEGF-A exists in six isoforms produced by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. These isoforms differ primarily in their bioavailability, with VEGF165 being the major isoform. VEGF is believed to be an important regulator of normal and disease-related angiogenesis.

In addition to the VEGF family, human angiogenin is thought to be involved in vascular development and postnatal angiogenesis. Human angiogenin includes the naturally-occurring agonist angiogenin-1 (ANG-1) and the naturally-occurring antagonist angiogenin-2 (ANG-2). The effect of ANG-1 is thought to be conserved in adults, where it is widely and constitutively expressed. In contrast, ANG-2 expression is primarily limited to sites of vascular remodeling where it is thought to block the constitutive stabilizing or maturation function of ANG-1, allowing the blood vessel to revert to and remain in a plastic state that may be more responsive to budding signals.

In recent years, sub>A number of bispecific antibodies have been developed that target VEGF-A and ANG-2 (e.g., WO2012131078 and WO 2014009465). However, existing bispecific antibodies have poor blocking properties against VEGF and Ang2 and, due to the too large molecular weight, result in lower molar concentrations upon single administration. In particular, for ocular diseases, smaller molecular weight antibodies are typically used by intravitreal application and require less frequency of administration. Thus, there remains Sub>A need for new bispecific binding molecules targeting VEGF-A and ANG-2, particularly for use in ocular diseases.

Disclosure of Invention

The present invention exploits novel VHH antibodies targeting VEGF-A or ANG-2, as well as bispecific binding molecules directed against VEGF-A and ANG2 simultaneously. In particular, the bispecific binding molecules of the invention have a lower molecular weight and a higher molar concentration at the same mass concentration than known antibodies; and has stronger VEGF A and Ang2 blocking activity, and can completely block the primary cell proliferation induced by VEGFA. Thus, the molecules of the invention have clinically stronger blocking activity, and can make the molar concentration of the antibody higher in single administration, maintain the efficacy of single administration for a longer time, and reduce the frequency of ocular administration (such as intravitreal injection).

Description of the drawings:

FIG. 1 shows the structure of a bispecific binding molecule.

Figure 2 shows that anti-VEGF a VHH antibodies assayed using ELISA were able to block VEGF a binding to VEGFR 2.

FIG. 3 shows the effect of humanized anti-Ang2 VHH antibodies blocking binding of Ang2 to Tie2 as determined using ELISA.

FIG. 4 shows the effect of anti-Ang2 VHH antibody (A) and humanized anti-Ang2 VHH antibody (B) on inhibition of hAN 2-Fc-induced phosphorylation of 293-Tie2 cells as determined by ELISA.

FIG. 5 shows the detection of the effect of anti-VEGF A VHH blocking VEGFA activation of KDR receptor using HEK293-KDR reporter method.

FIG. 6 shows the effect of CCK-8 assay anti-VEGF A VHH antibodies on inhibition of VEGF A induction of survival and proliferation of HUVEC cells.

FIG. 7 shows the effect of blocking VEGF activation of KDR receptor by VEGF A/Ang2 bispecific binding molecule IEX04-012 using the HEK293-KDR reporter method.

FIG. 8 shows the effect of bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 on inhibition of VEGF-induced HUVEC cell survival and proliferation.

FIG. 9 shows that the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 block binding of human Ang2 to Tie2 as determined by ELISA.

FIG. 10 shows that flow cytometry assays determine that bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 block binding of Ang2-Fc to Tie 2.

FIG. 11 shows that the bispecific binding molecule IEX04-012 of the invention effectively inhibits hAN 2-Fc-induced 293-Tie2 phosphorylation in vitro as measured using flow cytometry.

FIG. 12 shows that bispecific binding molecule IEX04-012 of the invention reduces VEGF-induced vascular endothelial cell permeability, i.e., inhibits VEGF-induced HUVEC cell leakage.

Figure 13 shows laser spot fraction statistics for grade 4 (panel a) and above grade 3 (panel B) in a laser induced choroidal neovascularization model.

Figure 14 shows the retinal membrane thickness statistics in a laser-induced choroidal neovascularization model.

FIG. 15 shows leakage area statistics in a laser-induced model of choroidal neovascularization

Fig. 16 shows a graph of H & E staining of ocular fundus tissue for the laser-induced choroidal neovascularization model (a) and lesion area statistics (B).

Figure 17 shows a graph of CD31 staining of fundus tissue for the laser-induced choroidal neovascularization model (a) and positive cell statistics (B).

Definition of the definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the selectable items or two or more or all of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

The term "VEGF" as used herein refers to vascular growth factor. VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) and endocrine gland derived VEGF (EG-VEGF). The active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members. VEGF-A exists in six isoforms produced by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. These isoforms differ primarily in their bioavailability, with VEGF165 being the major isoform. In some embodiments, VEGF A of the invention refers to VEGF A from a human, such as VEGF165 from a human. In one embodiment, the amino acid sequence of VEGFA of the invention is the amino acid sequence of accession number P15692 (uniprot database).

As used herein, the term "ANG2" refers to human angiopoietin-2 (ANG-2) (alternatively abbreviated as ANGPT2 or ANG 2), which is described, for example, in Maisonpierre, P.C. et al, science277 (1997) 55-60 and Cheung, A.H. et al, genomics 48 (1998) 389-91. Ang1 and Ang2 were found as ligands for the tyrosine kinase family Tie selectively expressed in vascular endothelium. There are currently 4 established angiopoietin family members. Angiopoietin-3 and-4 (ANG 3 and ANG 4) may represent a wide variety of counterparts of the same locus in mice and humans. Ang1 and Ang2 were initially identified as agonists and antagonists, respectively, in tissue culture experiments (for ANG1, see Davis, S. Et al., cell87 (1996) 1161-69; for ANG2, see Maisonpierre, P.C. et al., science277 (1997) 55-60). All known angiogenin binds predominantly to Tie 2. In some embodiments, ANG2 of the invention refers to ANG2 from a human. In some embodiments, human Ang2 comprises the amino acid sequence of accession No. O15123 (uniprot database).

The term "multispecific binding molecule" refers to a multispecific binding molecule, e.g., bispecific binding molecule, that is, the molecule comprises at least a first target-binding region and a second target-binding region, wherein the first target-binding region binds one target or antigen and the second target-binding region binds another antigen or target. Thus, a molecule according to the invention comprises a specificity for at least two different antigens or targets. The molecules according to the invention also encompass multispecific molecules, such as trispecific binding molecules, comprising multiple target binding regions/binding sites. In some embodiments, the bispecific binding molecules of the invention are bispecific antibodies.

The term "linker" as used herein refers to any molecule that enables direct attachment of different parts of a bispecific binding molecule. Examples of linkers that establish covalent linkages between different molecular moieties include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide or copolymers of polyethylene glycol, polypropylene glycol. In some embodiments, the linker is a peptide linker, which refers to a sequence of amino acids, wherein the sequence links the amino acid sequence of the first portion of the binding molecule to the second portion of the binding molecule. For example, a peptide linker may connect a first target binding region of a binding molecule to a second target binding region. For example, a peptide linker may also connect one portion of an antibody to another portion of an antibody, such as connecting a light chain variable region to a heavy chain variable region. Preferably, the peptide linker has a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity.

The peptide linker may or may not include predominantly the following amino acid residues: gly, ser, ala or Thr. Useful linkers include glycine-serine polymers, including for example (GS) _n 、(GSGGS) _n 、(GGGGS) _n 、(GGGS) _n And (GGGGS) _n G, wherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Useful linkers also include glycine monomer polymerizations, e.g. (G) _n Wherein n is an integer of at least 4 (and for example 4-20, for example 4, 5, 6, 7, 8, 9, 10 and more). Preferably, the linker is (GGGGS) _n Where n=1, 2, 3 or 4.

The term "valency" according to the invention means the presence of a specified number of binding sites in a binding molecule, e.g. an antibody molecule. Thus, the terms bivalent, trivalent, tetravalent respectively denote the presence of two, three or four binding sites (target binding regions) in the binding molecule. Bispecific binding molecules according to the invention are at least divalent and may be multivalent, e.g. divalent, trivalent, tetravalent or hexavalent.

The term "target binding region" as used herein refers to any portion of a multispecific binding molecule, e.g., bispecific binding molecule, that binds a particular target or antigen. The target binding region may be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such target binding regions may or may not have a tertiary structure independent of the remainder of BsAB, and may or may not bind as separate entities to their targets. The target binding region may also be a receptor or ligand, or a domain of a receptor capable of binding a ligand.

The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen binding fragment.

An "antigen binding fragment" refers to a molecule that is different from an intact antibody, which comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ The method comprises the steps of carrying out a first treatment on the surface of the dAb (domain antibody); a linear antibody; single chain antibodies (e.g., scFv); single domain antibodies such as VHH; a diabody or fragment thereof; or camelidae antibodies.

"VHH", also known as a single domain antibody (single domain antibody, sdAb), refers to a genetically engineered antibody consisting of only heavy chain antibody Variable regions (Variable regions), which contains only 3 HCDRs of the heavy chain Variable regions. VHH has antigen specificity and high affinity with only 3 HCDRs, whereas common antibodies require 6 CDRs. The crystal structure shows that VHH is composed of 2 β -sheet scaffolds, similar to the conventional antibody VH immunoglobulin fold.

The term "target" refers to the bound object to which the binding molecule is directed. The target may be an antigen or may be a ligand or receptor.

The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may involve antibody production or activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including substantially all proteins or peptides, may be used as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen (e.g., VEGF or Ang 2) that specifically interacts with an antibody molecule.

"complementarity determining regions" or "CDR regions" or "CDRs" are regions of an antibody variable domain that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al, (1989) Nature342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273,927-948 (1997)), kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Pat. No. of Health and Human Services, national Institutes ofHealth (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (on the world Wide Web) based on antibody sequence variability, and North definition based on neighbor-transmitted clusters (CDR affinity propagation clustering) using a large number of crystal structures.

For example, the residues of each CDR are as follows, according to different CDR determination schemes.

CDRs may also be determined based on having the same Kabat numbering positions as the reference CDR sequences (e.g., any of the exemplary CDRs of the invention).

In the present invention, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways.

In the present invention, unless otherwise indicated, when referring to residue positions in the antibody variable region, including heavy chain variable region residues and light chain variable region residues, reference is made to numbering positions according to the Kabat numbering system (Kabat et al Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md. (1991)).

In one embodiment, the CDRs in the VHH of the invention are according to the following rules: wherein HCDR1 is determined according to AbM and HCDR2 and HCDR3 are determined according to Kabat.

It should be noted that the boundaries of CDRs of variable regions of the same antibody obtained based on different assignment systems may differ. I.e. the CDR sequences of the same antibody variable region defined under different assignment systems are different. Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The minimum overlap region can be determined using at least two of the Kabat, chothia, abM, contact and North methods, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be apparent to those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure of the antibody and the protein folding. Thus, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues according to the Kabat or Chothia definition may be replaced by conserved amino acid residues.

The term "Fc region" is used herein to define the constant regions of CH2 and CH3 of an immunoglobulin heavy chain, and includes native sequence Fc regions and variant Fc regions. The native or wild-type Fc region is capable of binding to different Fc receptors on the surface of immune cells, thereby being capable of eliciting cdc\adcc\adcp effector function. Such effector functions typically require that the Fc region be associated with a binding domain (e.g., an antibody variable region). In some embodiments, the Fc region is mutated to enhance its cdc\adcc\adcp effector function. In some embodiments, the Fc region is mutated to impair or delete its cdc\adcc\adcp effector function.

"humanized" antibody refers to an antibody that comprises amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. "human antibody" or "fully human antibody" may be used interchangeably to refer to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.

As used herein, the term "anti," "binding" or "specific binding" means that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of the binding site to bind to a particular target or antigen may be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by Radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or Surface Plasmon Resonance (SPR).

The term "effective amount" refers to an amount or dose of an antibody or fragment or composition or combination of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prevention.

"therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are less than the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100% relative to an untreated subject.

"prophylactically effective amount" means an amount effective to achieve the desired prophylactic result at the desired dosage and for the desired period of time. Typically, since the prophylactic dose is administered in the subject prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny selected or selected for the same function or biological activity in the initially transformed cells.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to and facilitates detection of an agent (such as a polynucleotide probe or antibody) to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels) or in the case of enzymatic labels may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. In some embodiments, the label is hFc or biotin.

"individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody or molecule is an antibody or molecule that has been separated from components of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

Calculation of sequence identity between sequences was performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequences. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparison and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) j.mol.biol.48:444-453) algorithm (available at http:// www.gcg.com) which has been integrated into the GAP program of the GCG software package, using the Blossum62 matrix or PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com) using the NWS gapdna.CMP matrix and the GAP weights 40, 50, 60, 70 or 80 and the length weights 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise indicated) is the Blossum62 scoring matrix employing gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5. The percent identity between two amino acid sequences or nucleotide sequences can also be determined using PAM120 weighted remainder table, gap length penalty 12, gap penalty 4) using the e.meyers and w.miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17). Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

The "ocular disease" as referred to herein encompasses ocular diseases involving angiogenesis (e.g., diseases occurring in the eye), such as those associated with corneal neovascularization.

The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. for administration with the active substance.

The term "pharmaceutical composition" refers to a composition that exists in a form that is effective to allow the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administered.

As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

"subject/patient/individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, like an organ or tissue sample or biopsy or puncture sample from fresh, frozen and/or preserved; blood or any blood component; body fluids such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of gestation or development of a subject. In some embodiments, the tissue sample is ocular tissue, such as vitreous. In some embodiments, the sample is tear fluid or vitreous humor. Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

detailed description of the preferred embodiments

I. VHH antibodies against VEGF A

The present invention relates to VHH antibodies directed against VEGF a. In some embodiments, the anti-VEGF VHH of the invention comprises 3 CDRs, HCDR1, HCDR2 and HCDR3, wherein

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 1;

HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 2;

HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 3;

or alternatively

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 6;

HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 7 or 10;

HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 8.

In some embodiments, the VHH comprises or consists of an amino acid sequence set forth in SEQ ID NO. 4, 5, 9 or 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth in SEQ ID NO. 4, 5, 9 or 11.

In some embodiments, the VHH comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO. 4, 5, 9 or 11, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.

In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2, or HCDR 3.

VHH antibodies against Ang2

The present invention relates to VHH antibodies against Ang 2. In some embodiments, the anti-Ang 2 VHH of the invention comprises 3 CDRs, HCDR1, HCDR2 and HCDR3, wherein

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 16;

HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 17 or 20;

HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 18.

In some embodiments, the VHH comprises or consists of an amino acid sequence set forth in SEQ ID NO. 19 or 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 19 or 21.

In some embodiments, the VHH comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO. 19 or 21, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.

Bispecific binding molecules against VEGF A x ANG2

In one aspect of the invention, the invention relates to a bispecific binding molecule that binds VEGF a and Ang2, comprising a first target binding domain that specifically binds VEGF a and a second target binding domain that specifically binds Ang2, wherein the second target binding domain is a VHH against Ang2, e.g., an anti-Ang 2 VHH of the invention as described above.

In some embodiments, the primary target binding region is selected from the group consisting of

VHH that specifically binds VEGF a;

an antigen binding fragment of an antibody that specifically binds VEGF a, e.g., an scFv, e.g., the antibody is a fully human antibody or a humanized antibody; or (b)

VEGF receptor (VEGF R) or an extracellular domain thereof or a fusion protein comprising an extracellular domain thereof, e.g. a fusion protein of an extracellular domain with Fc, which specifically binds to VEGF a.

In some embodiments, a bispecific binding molecule of the invention comprises 1 or 2 or 3 or 4 primary or secondary target binding regions. In some embodiments, a bispecific binding molecule of the invention comprises 2, 3, or 4 target binding regions. In some embodiments, the bispecific binding molecules of the invention are bivalent or 3-valent or 4-valent. In some embodiments, the bispecific binding molecules of the invention are bispecific antibodies.

In one aspect of the invention, a bispecific binding molecule of the invention, e.g., a bispecific antibody, has the following structure:

light chain variable region of anti-VEGF antibody VL-linker-heavy chain variable region of anti-VEGF antibody VH-linker-anti-Ang 2 VHH or

Heavy chain variable region of anti-VEGF antibody VH-linker-anti-Ang 2 VHH;

wherein the anti-Ang 2 VHH comprises or consists of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO. 16; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 17 or 20; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 18.

In some embodiments, the structure of the bispecific binding molecules described above is shown in fig. 1A or fig. 1B. In some embodiments, the bispecific binding molecule consists of one chain. In some embodiments, the bispecific binding molecules described above are bivalent.

In some embodiments, the light chain variable region VL of an anti-VEGF antibody comprises or consists of LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence set forth in SEQ ID NO. 31; LCDR2 comprises or consists of the sequence shown in SEQ ID NO. 32; LCDR3 comprises or consists of the sequence shown in SEQ ID NO. 33.

In some embodiments, the heavy chain variable region VH of the anti-VEGF antibody comprises or consists of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID No. 35; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 36; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 37.

In some embodiments, the heavy chain variable region VH of an anti-VEGF antibody of the invention comprises, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 34. In some embodiments, the heavy chain variable region VH comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID No. 34, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2, or HCDR 3.

In some embodiments, the light chain variable region VL of an anti-VEGF antibody of the invention comprises or consists of an amino acid sequence set forth in SEQ ID NO. 30, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 30. In some embodiments, the light chain variable region VL comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO. 30. In some preferred embodiments, the mutation is not present in a CDR, such as LCDR1, LCDR2, or LCDR 3.

In some embodiments, an anti-Ang 2 VHH of the invention comprises, or comprises, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence described in SEQ ID No. 19 or 21, or consists of an amino acid described in SEQ ID No. 19 or 21. In some embodiments, the VHH comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO. 19 or 21, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2, or HCDR 3.

In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO. 23. In some embodiments, the linker between the light chain variable region and the heavy chain variable region of the anti-VEGF antibody comprises or consists of the amino acid sequence of SEQ ID NO:23, wherein, for example, n=4. In some embodiments, the linker between the anti-VEGF variable region and the anti-Ang 2 VHH comprises or consists of the amino acid sequence of SEQ ID No. 23, wherein, for example, n=2 or 3, for example 3.

In some embodiments, the bispecific binding molecules of the invention against VEGF A x ANG2 comprise, or consist of, the amino acid sequence of SEQ ID NO. 28, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 28. In some embodiments, the bispecific binding molecule comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID No. 28, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in the anti-VEGF antibody variable region and in the CDRs against Ang2 VHH.

In another aspect of the invention, a bispecific binding molecule of the invention, e.g., a bispecific antibody, has the following structure:

a first anti-VEGF VHH-linker-second anti-VEGF VHH-linker-anti-Ang 2 VHH,

In some embodiments, the structure of the bispecific binding molecules described above is shown in figure 1C. In some embodiments, the bispecific binding molecule consists of one chain. In some embodiments, the bispecific binding molecules described above are trivalent. In some embodiments, the first anti-VEGF VHH is the same as or different from the second anti-VEGF VHH.

In some embodiments, the anti-VEGF VHH comprises or consists of HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence set forth in SEQ ID NO. 1; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 2; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 3;

or alternatively

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 6; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 7 or 10; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 8.

In some embodiments, the anti-VEGF VHH comprises or consists of an amino acid sequence set forth in SEQ ID NO. 4, 5, 9 or 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth in SEQ ID NO. 4, 5, 9 or 11. In some embodiments, the VHH comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO. 4, 5, 9 or 11, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2, or HCDR 3.

In some embodiments, the anti-VEGF VHH comprises or consists of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence set forth in SEQ ID NO. 6; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 7 or 10; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 8.

In some embodiments, the anti-VEGF VHH comprises or consists of an amino acid sequence set forth in SEQ ID NO 9 or 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO 9 or 11. In some embodiments, the VHH comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO. 9 or 11, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2, or HCDR 3.

In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO. 23. In some embodiments, the linker between the first anti-VEGF VHH and the second anti-VEGF VHH or the linker between the second anti-VEGF VHH and the anti-Ang 2 VHH comprises or consists of the amino acid sequence of SEQ ID No. 23, e.g., n=2.

In some embodiments, the bispecific binding molecules of the invention against VEGF A x ANG2 comprise, or consist of, the amino acid sequence of SEQ ID NO. 22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 22. In some embodiments, the bispecific binding molecule comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID No. 22, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in the CDRs against VEGF VHH and anti-Ang 2 VHH.

In another aspect of the invention, the bispecific binding molecules of the invention comprise one or two of the following chains:

VEGF R ectodomain-Fc-linker-anti-Ang 2 VHH

In some embodiments, the structure of the bispecific binding molecules described above is shown in figure 1D. In some embodiments, the bispecific binding molecules described above consist of two chains. In some embodiments, the bispecific binding molecule is tetravalent.

In some embodiments, the anti-Ang 2 VHH comprises or consists of an amino acid sequence of SEQ ID No. 19 or 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID No. 19 or 21. In some embodiments, the VHH comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO. 19 or 21, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2, or HCDR 3.

In some embodiments, the VEGFR ectodomain is an ectodomain of VEGFR from a human. In some embodiments, the VEGFR extracellular domain comprises a VEGFR1 second antibody-like domain (e.g., FLT1 domain 2) and a VEGFR2 third antibody-like domain (e.g., KDR domain 3). In some embodiments, the VEGFR extracellular domain comprises a human VEGFR1 second antibody-like domain and a human VEGFR2 third antibody-like domain. In some embodiments, the VEGFR extracellular domain comprises or consists of the amino acid sequence of SEQ ID NO. 26, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 26. In some embodiments, the VEGFR ectodomain comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence set forth in SEQ ID No. 26, preferably the VEGFR ectodomain retains a binding affinity for VEGF similar to the domain set forth in SEQ ID No. 26 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

In some embodiments, the Fc is an Fc derived from human IgG1, igG2, igG3, or IgG4, such as a wild-type Fc, or an Fc variant known in the art. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO. 27, or comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 27.

In some embodiments, the VEGF R ectodomain-Fc is a fusion protein of a VEGFR ectodomain and Fc, e.g., afiibrecept or a derivative thereof.

In some embodiments, the VEGF R extracellular domain-Fc comprises or consists of the amino acid sequence of SEQ ID NO. 25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 25. In some embodiments, the VEGF R ectodomain-Fc comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO. 25, preferably the VEGF R ectodomain-Fc retains binding affinity to VEGF similar to the domain set forth in SEQ ID NO. 25 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID No. 23, e.g., n=3.

In some embodiments, one strand of the bispecific binding molecule against VEGF A×ANG2 of the invention comprises the amino acid sequence of SEQ ID NO. 24, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 24, or consists of the amino acids set forth in SEQ ID NO. 24. In some embodiments, the bispecific binding molecule comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID No. 24, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions. In some preferred embodiments, the mutation is not present in a CDR of the anti-Ang 2 VHH. In some embodiments, the mutant VEGF R ectodomain-Fc retains binding affinity to VEGF similar to the domain shown in SEQ ID NO. 25 (e.g., has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

In one embodiment of the invention, an antibody or binding molecule described herein comprises one or more amino acid mutations. In some embodiments, the amino acid mutation comprises a substitution, insertion, or deletion of an amino acid. Preferably, the amino acids described herein are changed to amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid mutations described in the present invention occur in regions outside the CDRs (e.g., in the FR). In some embodiments, the amino acid mutations described herein occur in an antibody heavy chain constant region, e.g., an Fc region, and in preferred embodiments, the amino acid mutations in the Fc region impair or delete ADCC and/or CDC effects of the antibody.

In some embodiments, the substitutions are conservative substitutions. Conservative substitutions refer to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid.

In certain embodiments, one or more amino acid mutations can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, complement-dependent cytotoxicity, fc receptor binding, and/or antibody-dependent cytotoxicity. The Fc region variant may include a human Fc region sequence (e.g., a human IgGl, igG2, igG3, or IgG4 Fc region) comprising an amino acid mutation (e.g., substitution) at one or more amino acid positions.

In certain embodiments, it may be desirable to mutate the variable region of an antibody to generate disulfide bonds, e.g., to generate an scFv comprising disulfide bond mutations.

In certain embodiments, the antibodies or binding molecules provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Moieties suitable for such derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

Properties of the VHH antibodies or bispecific binding molecules of the invention

In some embodiments, the anti-Ang 2 VHH antibodies of the invention are capable of specifically binding Ang2, e.g., human Ang2, e.g., with high affinity.

In some embodiments, the anti-VEGFA VHH antibodies of the invention are capable of specifically binding to VEGF a, e.g., human VEGF a, e.g., with high affinity.

In some embodiments, the bispecific binding molecules of the invention are capable of specifically binding Ang2 and VEGFA, e.g., human Ang2 and human VEGFA, e.g., with high affinity.

In some embodiments, the anti-Ang 2 antibody or anti-VEGFA antibody or bispecific binding molecule of the invention has one or more of the following properties:

(i) The anti-Ang 2 antibody or the bispecific binding molecule has an inhibitory effect on Ang 2-induced Tie2 phosphorylation;

(ii) An anti-Ang 2 antibody or bispecific binding molecule has a blocking effect on binding between Ang2 and Tie 2;

(iii) anti-VEGFA antibodies or bispecific binding molecules have a blocking effect on VEGFA activation-related receptor signaling pathways, e.g., as detected by KDR reporter;

(iv) The anti-VEGFA antibody or bispecific binding molecule has a blocking effect on binding between VEGFA and VEGFR;

(v) The anti-VEGFA antibodies or bispecific binding molecules have an inhibitory effect on VEGF a-induced survival and proliferation of cells (e.g., primary cells, such as vascular endothelial cells, e.g., human umbilical vein endothelial cells, e.g., HUVECs);

(vi) Bispecific binding molecules have an inhibitory effect on vascular endothelial cell (e.g., human umbilical vein endothelial cells, such as HUVEC) leakage;

(vii) Bispecific binding molecules have inhibitory effects on neovascularization, such as inhibition of ocular fundus or retinal choroidal neovascularization, e.g., inhibition of leakage from new blood vessels, e.g., preservation of vascular integrity, in vivo or in vitro.

V. nucleic acids of the invention and host cells comprising the same

In one aspect, the invention provides a nucleic acid encoding any of the VHH or bispecific binding molecules above or any one of the strands thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs). In one embodiment, the vector is, for example, pcDNA3.1. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell (e.g., CHO-S) or 293 cell (e.g., 293F or HEK293 cell)), or other cell suitable for the production of antibodies or fragments thereof. In another embodiment, the host cell is prokaryotic, e.g., E.coli, e.g., TG1.

For example, a nucleic acid of the invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs 4, 5, 9, 11, 19, 21, 22, 24, 28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs 4, 5, 9, 11, 19, 21, 22, 24, 28.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing vectors encoding VHH or bispecific binding molecules include prokaryotic or eukaryotic cells as described herein. For example, VHH may be produced in bacteria. After expression, VHH is present in the culture supernatant and may be further purified.

In one embodiment, the host cell is prokaryotic, e.g., is a bacterium, e.g., E.coli, e.g., TG1.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for the preparation of antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with a partially or fully human glycosylation pattern. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV 40; human embryonic kidney (HEK 293, 293F or 293T cells), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, expi CHO, and the like; and myeloma cell lines such as Y0, NS0 and Sp2/0. Mammalian host cell lines suitable for antibody production are known in the art.

Production and purification of VHH or bispecific binding molecules of the invention

In one embodiment, a method of preparing a VHH or bispecific binding molecule of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the VHH or bispecific binding molecule (e.g. any one polypeptide chain and/or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for expression of the VHH or bispecific binding molecule or a chain thereof, and optionally recovering the VHH or bispecific binding molecule from the host cell (or host cell culture medium).

For recombinant production of the molecules of the invention, nucleic acids encoding the VHH or bispecific binding molecules of the invention (e.g. the molecules described above, e.g. any one polypeptide chain and/or multiple polypeptide chains) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures.

VHH or bispecific binding molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention may be determined by any of a variety of well-known analytical methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

VII assay

The VHH or bispecific binding molecules provided herein can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the VHH or bispecific binding molecules of the invention are tested for their target (e.g. antigen) binding activity, for example by known methods such as biofilm thin layer interference techniques, ELISA, etc. Binding to VEGF A and/or Ang2 may be determined using methods known in the art, exemplary methods being disclosed herein. In some embodiments, radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or Surface Plasmon Resonance (SPR) or flow cytometry measurement is used.

The invention also provides assays for identifying VHH or bispecific binding molecules that are biologically active. The biological activity is selected from, for example

(i) Inhibition of Ang 2-induced Tie2 phosphorylation by anti-Ang 2 antibodies or bispecific binding molecules;

(ii) Blocking of binding between Ang2 and Tie2 by anti-Ang 2 antibodies or bispecific binding molecules;

(iii) Blocking of VEGFA-activating receptor signaling pathway by anti-VEGFA antibodies or bispecific binding molecules, e.g., as detected by KDR reporter;

(iv) Blocking of binding between VEGFA and VEGFR by anti-VEGFA antibodies or bispecific binding molecules;

(iv) Inhibition of VEGF a-induced survival and proliferation of cells (e.g., primary cells, e.g., vascular endothelial cells, e.g., human umbilical vein endothelial cells, e.g., HUVEC) by anti-VEGFA antibodies or bispecific binding molecules;

(vi) Inhibition of vascular endothelial cell (e.g., human umbilical vein endothelial cells, such as HUVEC) leakage by bispecific binding molecules;

(vii) The bispecific binding molecules have an inhibitory effect on neovascularization, such as inhibition of ocular fundus or retinochoroidal neovascularization, e.g., inhibition of neovascular-induced leakage, e.g., preservation of vascular integrity, in vivo or in vitro.

Cells for use in any of the in vitro assays described above are primary cells or cell lines, including cells that naturally express or overexpress the Ang2 receptor (e.g., tie 2) or VEGFR (e.g., VEGFR2, KDR), e.g., 293 cells that overexpress Tie2 or KDR, e.g., HEK293 or Expi293; or vascular endothelial cells, such as human umbilical vein endothelial cells, e.g., HUVEC.

In some embodiments, the assay may be performed using a label such as biotin or hFc.

It will be appreciated that any of the above assays can be performed using the combination of the antibodies of the invention and other active agents.

Immunoconjugates and pharmaceutical compositions

In some embodiments, the invention provides immunoconjugates comprising any VHH or bispecific binding molecule described herein. Preferably, the immunoconjugate comprises one or more other therapeutic agents or labels.

In some embodiments, the invention provides a composition or medicament or formulation comprising any VHH or bispecific binding molecule described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, e.g., a pharmaceutical composition, comprises a VHH or bispecific binding molecule of the invention in combination with one or more other therapeutic agents.

The invention also includes compositions (including pharmaceutical compositions) or medicaments or formulations comprising a VHH or bispecific binding molecule of the invention. These compositions or medicaments or formulations may also comprise suitable pharmaceutical excipients, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients as known in the art, including buffers.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

For the use of pharmaceutical excipients and their use, see also "Handbook of Pharmaceutical Excipients", eighth edition, R.C.Rowe, P.J.Seskey and s.c. owen, pharmaceutical Press, london, chicago.

The composition or medicament or formulation of the present invention may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions or eye drops), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and the therapeutic use. For example, the composition or medicament or formulation of the invention may be an eye drop.

The medicament or formulation comprising a VHH or bispecific binding molecule of the invention may be prepared by mixing a VHH or bispecific binding molecule of the invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

The compositions or medicaments or formulations of the invention may also contain more than one active ingredient which is required for the particular indication being treated, preferably those active ingredients which have complementary activities which do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

The invention also provides a pharmaceutical combination or pharmaceutical combination product comprising a VHH or bispecific binding molecule of the invention, and one or more other therapeutic agents. The invention also provides kits comprising the pharmaceutical combinations, e.g., the kits comprising, in the same package:

-a first container containing a pharmaceutical composition comprising a VHH or bispecific binding molecule of the invention;

-a second container containing a pharmaceutical composition comprising the other therapeutic agent.

IX. uses and methods

In one aspect, the invention provides a method of preventing or treating an ocular disease in a subject comprising administering to the subject an effective amount of an anti-VEGF VHH of the invention, an anti-Ang 2 VHH of the invention, or a bispecific binding molecule of the invention, or a composition or a medicament or formulation comprising the same.

In some embodiments, the patient has (e.g., elevated levels of, e.g., nucleic acid or protein levels of) VEGF, e.g., VEGF a, and/or Ang2 therein.

In some embodiments, the ocular disease includes, but is not limited to, ocular diseases associated with angiogenesis, such as ocular diseases associated with corneal neovascularization.

In some embodiments, the ocular disease treatment will benefit from inhibition of nucleic acid or protein levels of VEGF, e.g., VEGF a, and/or Ang2.

In other aspects, the invention provides the use of an anti-VEGF VHH of the invention, an anti-Ang 2 VHH of the invention or a bispecific binding molecule of the invention or a composition comprising the same in the manufacture or preparation of a medicament for use as described herein, e.g. for the prevention or treatment of a related disease or disorder as referred to herein.

In some embodiments, an anti-VEGF VHH of the invention, an anti-Ang 2 VHH of the invention, or a bispecific binding molecule of the invention, or a composition or a pharmaceutical or formulation comprising the same, delays the onset of a disorder and/or symptoms associated with the disorder.

In some embodiments, an anti-VEGF VHH of the invention, an anti-Ang 2 VHH of the invention, or a composition or a medicament or formulation comprising the same, can also be administered in combination with one or more other therapies, e.g., therapeutic modalities and/or other therapeutic agents, for use described herein, e.g., for preventing and/or treating a related disease or disorder mentioned herein.

The route of administration of the anti-VEGF VHH of the invention, the anti-Ang 2 VHH of the invention or the bispecific binding molecule of the invention or the composition or medicament or formulation comprising the same is according to known methods, e.g. topical administration, e.g. intraocular administration, ocular surface administration. In some embodiments, administration is by injection or instillation.

Methods and compositions for diagnosis and detection

In certain embodiments, the anti-Ang 2 VHH antibodies provided herein can be used to detect the presence of Ang2 in a biological sample. In certain embodiments, the anti-VEGFA VHH antibodies provided herein can be used to detect the presence of VEGFA in a biological sample. In certain embodiments, the anti-bispecific binding molecules provided herein can be used to detect the presence of Ang2 and/or VEGFA in a biological sample.

The term "detection" as used herein, including quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a bodily fluid or ocular tissue, such as a vitreous humor.

In certain embodiments, the method comprises contacting a biological sample with a VHH or bispecific binding molecule as described herein under conditions that allow it to bind to Ang2 or VEGFA, and detecting whether a complex is formed between the VHH or bispecific binding molecule and Ang2 or VEGFA. The formation of a complex indicates the presence of Ang2 or VEGFA. The method may be an in vitro or an in vivo method. In one embodiment, the antibodies of the invention are used to select a subject suitable for treatment with a VHH or bispecific binding molecule of the invention, e.g., wherein Ang2 or VEGFA is a biomarker for selecting the subject.

In certain embodiments, a labeled VHH or bispecific binding molecule is provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radiolabels), as well as moieties that are detected indirectly, such as enzymes or ligands, e.g., by enzymatic reactions or molecular interactions. In some embodiments, the label is a label such as biotin or hFc.

In some embodiments provided herein, the sample is obtained prior to treatment with a VHH or bispecific binding molecule of the invention. In some embodiments, the sample is obtained prior to use with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

In some embodiments, ang2 and/or VEGF a is detected prior to treatment, e.g., prior to initiation of treatment or prior to a certain treatment following a treatment interval.

In some embodiments, there is provided a method of treating a disease of the invention, the method comprising: assaying a subject (e.g., a sample) (e.g., a subject sample) for the presence of Ang2 and/or VEGF a, thereby determining an Ang2 and/or VEGF a value, comparing the Ang2 and/or VEGF a value to a control value (e.g., a value in a normal individual), and if the Ang2 and/or VEGF a value is greater than the control value, administering to the subject a therapeutically effective amount of a VHH or bispecific binding molecule of the invention, optionally in combination with one or more other therapies, thereby treating the disease.

These and other aspects and embodiments of the invention are described in and exemplified by the following examples in the figures (which are briefly described to follow) and in the following detailed description. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention, however, it is to be understood that the examples are presented by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Example 1 preparation of phage immune library

Alpaca immunization or synthesis library construction

1.1 selecting 2 healthy adult alpacas (Chengdu Abak) and uniformly mixing 0.5mg of recombinant protein antigen VEGFA or Ang2 (Beijing Yiqiao) with Freund's adjuvant according to a ratio of 1:1, and immunizing the alpacas by subcutaneous multipoint injection at the back for four times at an immunization interval of 2 weeks.

1.2 collecting 50ml alpaca peripheral blood, isolating lymphocytes at a rate of every 2.5X10 ⁷ 1mL of Trizol reagent was added to each living cell, and total RNA was extracted by chloroform/isopropanol precipitation. 10ug of RNA was used as a template and reverse transcription was performed using PrimeScript reverse transcription kit (Takara Co.). The cDNA was used as a template for a first round of PCR reaction using the forward primer Alp-VhL and the reverse primer Alp-2b/2cR to obtain a first round of PCR product. The first round PCR product was used as a template for a second round PCR reaction using the forward primer Alp-VhF and the reverse primer Alp-JHR-SalI to obtain a second round PCR product. The pC3-HF vector and the second round PCR product are subjected to double digestion by using SacI and SalI (Thermo company), the digested products are added into T4 ligase (Thermo company) for reaction, TG1 competent cells are electrically transformed, a VHH antibody library is constructed, and bacterial liquid is frozen at-80 ℃.

The resuscitated bacterial solution was inoculated into 100ml YT-AG medium (Shanghai Biotechnology Co.), M13KO7 helper phage was added to infect, and the bacterial cells were resuspended in 2 XYT-AK medium (Shanghai Biotechnology Co.), and cultured overnight at 37℃at 200 rpm. The culture supernatant was collected and recombinant phage was prepared by PEG/NaCl precipitation.

1.3 recombinant phages were subjected to 3 rounds of panning experiments using the biotin-labeled antigen VEGFA (ACRO Co.) or Ang2 (Beijing Yiqiao Co.). 50ul of M280 magnetic beads (Thermo Co.) and the appropriate amount of biotin-labeled antigen were added to each tube, and after incubation for 30 minutes at room temperature, 1X10 was added ¹² cfu recombinant phage were incubated for 1 hour at room temperature. The resulting mixture was washed 10 times with 1ml of PBST for 5 minutes. Finally, 0.5ml of glycine buffer, pH2.5, was added, the antigen-bound recombinant phage was eluted, TG1 was infected overnight for culture, recombinant phage were prepared, used for the next round of panning experiments and TG1 bacterial clones of positive VHH were identified.

1.4 Binding ELISA detects Binding activity and clone sequencing.

VEGF A, ang2 antigen (Beijing Yiqiao) was pre-diluted to 0.5ug/ml with PBS buffer and coated onto 96-well ELISA plates, refrigerator overnight at 4 ℃. Antigen coated plates were washed 3 times with PBST, blocking agent was added to 300 ul/well, and left to stand at room temperature for blocking for 1 hour. PBST was washed 3 times, and 80ul of blocking agent+20 ul of expression supernatant of TG1 strain of positive VHH identified in 1.3 above was added, and the mixture was shaken at room temperature for 1 hour.

PBST was washed 3 times, 100 ul/well of Anti-Flag/HRP secondary antibody (Sigma Co.) diluted with blocking agent was added, and the mixture was shaken at room temperature for 40 minutes. PBST is washed for 6 times, TMB color development liquid is added to 100 ul/hole, and color development is carried out in dark place for 5-15 minutes. 100 ul/Kong Zhongzhi of liquid was added. Reading by an enzyme-labeled instrument, measuring an OD450nm absorbance value, selecting bacterial clones with a reading value greater than 0.5, carrying out gold-sending intelligent sequencing, selecting TG1 bacterial clones containing each corresponding VHH sequence, adding glycerol, and freezing in a refrigerator at-80 ℃.

EXAMPLE 2 production and purification of prokaryotic antibodies and humanization

The invention utilizes molecular biology technology to obtain antibody sequence in anti-VEGFA or Ang2 positive phage, and utilizes TG1 monoclonal expression containing positive VHH obtained as above to purify and obtain VHH antibody protein.

TG1 strain containing VHH expression plasmid identified in example 1 was inoculated into 800ml LB-Amp culture medium, and cultured at 37℃and 200rpm to OD600 value of 0.5-0.6. The bacterial solution was added with 1mM IPTG to induce expression, cultured overnight at 28℃at 200rpm, the culture supernatant was collected, centrifuged, 15ml PB+1mg/ml polymyxin was added to resuspend the bacteria, centrifuged again, and filtered through a 0.22um filter membrane. The bacterial lysate was passed through a 1ml Ni Sepharose preliminary column, washed 2 times with PBS, eluted the target protein with 0.5M imidazole, and the protein concentration was determined by UV. The eluted target protein is measured by ultraviolet method to obtain protein concentration, and the separated and packed multi-tube is stored in-40 deg.c refrigerator. The antibody solution obtained is subsequently referred to as supernatant.

The amino acid sequences of the CDRs and VHHs of the 2 anti-VEGF A VHH antibodies (LA 42F8 and LA46E 11) and the 1 anti-Ang 2 VHH antibodies (LA 24C 11) obtained by the invention, and the sequence numbers refer to a sequence table.

The immune repertoire antibodies LA42F8, LA46E11, LA24C11 obtained above were then humanized as follows:

(1) determining a CDR loop structure;

(2) finding the closest homologous sequence in each V/J region of the heavy chain in the human germline sequence database;

(3) screening the human germline that best matches the heavy chain light chain for a minimal amount of back mutations;

(4) constructing the CDR regions of the chimeric antibody onto human framework regions;

(5) determining amino acid positions in the framework region that function to maintain the CDR using the sequence and structural features;

(6) back-mutating (back to the input amino acid type) at sequence positions determined to be important;

(7) optimizing the amino acids of the risk site.

The amino acid sequences of the CDR and heavy chain variable region of the humanized VHH antibody LA42F8.5, LA46E11.8, LA24C11.10 obtained by the 3 humanized antibodies obtained by the invention are shown in the attached sequence table.

Expression of LA42F8, LA46E11, LA24C11 and humanized antibody LA42F8.5, LA46E11.8, LA24C11.10 in eukaryotic cells as described above was prepared as follows:

The humanized antibody sequences obtained from the above were cloned into pcdna3.1 (Invitrogen), and plasmids containing the antibody sequences were obtained, respectively.

Expi-293 cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to the one day prior to transfection1.5×10 ⁶ Individual cells/ml. Cell density on day of transfection was approximately 3X 10 ⁶ Individual cells/ml. 1/10 of the final volume of F17 medium (Gibco, A13835-01) was used as transfection buffer and the plasmid was added and mixed well. Appropriate Polyethylenimine (PEI) (Polysciences, 23966) was added to the plasmid (plasmid to PEI ratio 1:3 in 293F cells), mixed and incubated at room temperature for 10min to obtain a DNA/PEI mixture. After resuspension of the cells with the DNA/PEI mixture, 36.5℃and 8% CO ₂ . After 24h, 2% of the transfection volume of FEED (Sigma) was added, at 36.5℃and 120rpm,8% CO ₂ Culturing under the condition. And (3) continuously culturing until the 6 th day or the cell activity is less than or equal to 60%, and collecting and purifying cell supernatant.

The purification column was obtained by subjecting the gravity column to overnight treatment with 0.5M NaOH, washing with distilled water, and then drying at 180℃for 4 hours. The above collected cell supernatant was centrifuged at 4500rpm for 30min before purification, and the cells were discarded. The supernatant was filtered using a 0.22. Mu.l filter. Protein A column (Hitrap Mabselect Sure X5 ml, GE, 11-0034-95) was equilibrated with 10ml of binding buffer (sodium phosphate 20mM. NaCl150mM, PH7.0). The filtered supernatant was applied to a purification column and re-equilibrated with 15ml of binding buffer. 5ml of elution buffer (citric acid+sodium citrate 0.1M, pH 3.5) was added, and the eluate was collected, and 80. Mu.l of Tris-HCl was added per 1ml of eluate. The collected antibodies were ultrafiltration concentrated and exchanged into PBS (Gibco, 70011-044) and the concentration was measured. Except for the antibodies of the invention for detection in Table 3, FIG. 2, FIG. 4A and FIG. 5A, the following examples used the antibodies to which purified antibodies were expressed unless specifically mentioned as supernatants.

Similarly, nucleic acids encoding negative control IgG, positive control BI-anti-VEGF, BI836880, faricimab (see sequence listing) were cloned into pcDNA3.1, transfected into Expi-293 cells, expressed and purified to obtain the same procedures as in example 2.

EXAMPLE 3 determination of the kinetics of binding of chimeric antibodies of the invention to antigen by thin layer interference techniques of biological films

The equilibrium dissociation constant (KD) of the antibodies of the invention to human Ang2 was determined using biofilm thin layer interferometry (ForteBio). ForteBio affinity assays were performed according to the prior art (Estep, P et al, high throughput solution Based measurement of antibody-antigen affinity and epitope binding. MAbs,2013.5 (2): pages 270-8).

Half an hour before the start of the experiment, an appropriate number of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were taken based on the number of samples and immersed in SD buffer (PBS 1×, BSA 0.1%, tween-200.05%).

100 μl SD buffer, VHH antibody prepared in example 2 above, antigen [ including human Ang2 (Beijing Yiqiao), and human VEGF165 (R)&D)]Was added to 96-well black polystyrene half-cell plates (Greiner, 675076), respectively. The sensor positions are selected according to the sample position layout. The instrument set parameters are as follows: the operation steps are as follows: baseline, loading-1 nm, baseline, association and Dissociation; the run time of each step was dependent on the sample binding and dissociation speed, with a rotation speed of 400rpm and a temperature of 30 ℃. Analysis of K using ForteBio analysis software _D Values.

In the experiments described in the above assays, the affinities of the antibodies are shown in table 1:

table 1 affinity constants (equilibrium dissociation constants) for monovalent binding of fortebio detection antigen-antibody

ND represents undetected

TABLE 2 affinity constants (equilibrium dissociation constants) for the detection of bivalent binding of antigen-antibody by fortebio

^* Representing dissociation constants exceeding the ForteBio detection limit

EXAMPLE 4 ELISA blocking experiments with anti-VEGF A VHH antibodies

This example demonstrates the blocking effect of the anti-VEGF a VHH of the invention on hVEGF a binding to receptor KDR. SA (Thermo cat. No. 21125) was diluted to 1ug/ml and 100 ul/well was plated in enzyme-labeled plate overnight at 4 ℃. PBST was washed 3 times and blocked with 3% BSA for 1.5h. PBST was washed 3 times, 50ng/ml biotin-labeled VEGF A165 (ACRO cat. No. VE 5-H8210) was added, incubated for 1.5H, and the LA42F8 supernatant, LA46E11 supernatant and negative control IgG prepared in example 2 of the antibody (initial concentration 150ug/ml, 3-fold serial dilutions), and positive control BI-anti-VEGF) were pre-incubated with 50ul of VEGFR-Fc (Beijing Yinqiao, cat. No.: final concentration of 10012-H02H 0.2 ug/ml) was added to the plates after 20min incubation. PBST was washed 3 times, and incubated with anti-human Fc HRP antibody (Bethy cat# A80-104P) (1:10000) for 30min. PBST was washed 6 times, TMB developed for 5min, OD450nm reading after termination.

The blocking results of the 3 anti-VEGF VHH antibodies obtained by the invention are shown in FIG. 2. Fig. 2 shows that candidate molecule LA42F8, LA46E11 antibody, was able to completely block VEGF a binding to VEGFR 2.

EXAMPLE 5 ELISA blocking experiments with anti-Ang 2 VHH antibodies

This example demonstrates the blocking effect of the antibodies of the invention against Ang2 VHH on the binding of hAN 2-biotin (R & D cat# BT 623B/CF) to Tie2 protein

Tie2-Fc (Beijing Yiqiao brand number: 10700-H03H) was diluted to 2ug/ml and 100 ul/well was plated in an enzyme-labeled plate overnight at 4 ℃. PBST was washed 3 times and blocked with 3% BSA for 1.5h. PBST was washed 3 times, and 50ul of each of the purified LA24C11.10 and negative control IgG prepared in example 2 was incubated with hAN 2-biotin (final concentration 0.2 ug/ml) for 20min before addition to the plate. PBST was washed 3 times, incubated with Avidin HRP (1:2000) for 35min. PBST was washed 6 times, TMB developed for 5min, OD450nm reading after termination.

The blocking result of the anti-Ang 2 VHH antibody obtained by the invention is shown in Table 3, and the candidate molecule LA24C11 has blocking effect on the binding of Ang2 to the receptor Tie 2.

TABLE 3 Single Point blocking ELISA of purified antibodies

¹ Antibodies refer to the purified prokaryotic supernatant prepared in example 2, starting at a concentration of 0.556mg/ml, followed by dilution at 1:10 or 1:100;

² supernatant refers to the unpurified prokaryotic supernatant.

Similar to example 4 (using Ang2-Bio (R & D cat. Number: BT 623B/CF), the effect of LA24C11.10 blocking binding of Ang2 to Tie2 was examined by ELISA blocking experiments and the results are shown in FIG. 3. It can be seen that LA24C11.10 has a blocking effect on Ang2 binding at the receptor Tie 2.

EXAMPLE 6 phosphorylation assay of anti-Ang 2 VHH antibodies

This example demonstrates the inhibitory effect of the antibodies of the invention against Ang2 VHH on Tie2 phosphorylation induced by hang2-Fc.

hAN 2-induced phosphorylation assay

The present study incubates the antibody and recombinant hAN 2-Fc protein together with the Tie 2-overexpressing Expi293 cells 293-Tie2, and the content of phosphorylated Tie2 in the system is detected, so that the inhibition effect of different antibodies on the hAN 2-Fc induced Tie2 phosphorylation is reflected.

The human Tie2 overexpressing Expi-293 cells 293-Tie2 cells were generated by transfecting the Expi-293 cells (Thermo) with pCHO1.0 vector (Invitrogen) carrying the human Tie2 gene cloned into the multiple cloning site MCS (Beijing Yinqiao accession number: HG 10700-M).

293-Tie2 cells overexpressing human Tie2 were diluted to 2X 10 ⁶ Cells/ml, 100ul per well, were added to 96-well plates, centrifuged at 400g for 5min, and the supernatant removed.

The experimental medium was configured using an Expi293 medium (Thermo cat No. a 1435102): the test antibodies (LA 24C11 (24C 11) and LA24C11.0 (hz 24c11.10) prepared in example 2, as well as negative control IgG, and positive control Nesvacumab (prepared according to CN 202010573625.2)) were added at an initial final concentration of 60ug/ml, diluted in 1:2 equal ratios in sequence; the final concentration of hAN 2-Fc (Beijing Yiqiao: 10691-H02H) was 2.5ug/ml.

The cells were resuspended in 100ul of experimental medium per well, incubated at 37℃for 15min,

centrifuging to remove culture medium, adding 100ul NP-40 lysate (Biyun Tian, cat# P0013F) containing 1% protease (Thermo cat# 78442) and phosphatase inhibitor (Thermo cat# 78442), standing on ice for 30min, centrifuging 2000g, collecting protein supernatant, and storing in-80 deg.C refrigerator.

The concentration of pTie2 was measured according to the instructions of the phosphorylated Tie2ELISA kit (R & D cat# DYC 2720E) and the capture antibody in the kit was coated onto the ELISA plate at a concentration of 4ug/ml overnight at 4 ℃. PBST was washed three times and blocked with 5% bsa for 1h. 100ul of the protein supernatant obtained in the last step of freeze thawing was added, and a control pTie2 (R & D product number: DYC 2720E) was used to prepare a standard curve and incubated at room temperature for 2h (if the concentration of the sample pTie2 was too high, the obtained mixture could be diluted 2-to 3-fold beyond the ELISA detection range). PBST was washed three times, 100ul of HRP-conjugated anti-pTyr antibody (R & D cat# DYC 2720E) was added and incubated for 2h at room temperature. PBST was washed 6 times, developed by adding 100ul TMB (Solarbio cat# PR 1200), and after 15min the reaction was stopped by adding 100ul stop buffer (Solarbio cat# C1058). OD450-OD620 was measured using a multifunctional microplate reader SpectraMax i 3. The experimental results are shown in FIG. 4A (24C 11 is the LA24C11 prokaryotic expression purified supernatant prepared in example 2) and FIG. 4B.

Therefore, the anti-Ang 2 VHH antibodies LA24C11 and LA24C11.10 can effectively inhibit the 293-Tie2 cell phosphorylation induced by the hAN 2-Fc in vitro.

EXAMPLE 7 anti-VEGF A VHH antibody KDR reporter blocking assay

VEGF A can bind with a relevant receptor VEGFR2 (KDR), activate a VEGFR2 signal pathway, induce vascular endothelial cell survival, proliferation, migration and the like, and the blocking effect of a gradient diluted antibody on the VEGFA activation relevant receptor signal pathway is detected by using a KDR reporter experiment system and using NFAT-RE-luc2P/KDR HEK293 cells (Promega Cat CS 181401).

Experimental methods reference is made to the supplier (Promega) description:

NFAT-RE-luc2P/KDR HEK293 cells, which had been changed to experimental medium (DMEM medium with 10% fbs) 3 days in advance, were removed, old medium was aspirated, washed once with PBS, and then the cells were digested with 1ML Accutase solution (Sigma cat# a6964-500 ML),stopping the reaction until the cells become round and are wall-removed, sucking the cells into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding the culture medium, adding 10ml of dilution culture medium (DMEM culture medium containing 10% FBS) to resuspend the cells, uniformly mixing, and counting to obtain the cell viability which is above 90%. Cell density was adjusted to 0.8X10 with dilution medium ⁶ Mu.l/well of each/ml was added to 96-well white cell culture plates according to the experimental layout,

a mixture of VEGF A at a concentration of 100ng/ml and a gradient diluted antibody to be tested was prepared, allowed to stand for 30min, placed in a 96-well white cell culture plate containing cells at 50. Mu.l/well, and incubated in a 5% carbon dioxide incubator at 37℃for 6h. Wherein, the sample to be measured is as follows: negative control IgG; positive control BI-anti-VEGF; LA42F8; LA46E11; LA42F8.5; LA46E11.8; blank: only the dilution medium, no VEGF A, no antibody; VEGF A100ng/ml: only 100ng/ml VEGF A was contained.

Taking out the 96-well white cell culture plate incubated for 6 hours from the carbon dioxide incubator, and balancing for 10-15 min to room temperature. Bio-Glo Luciferase Assay System, which was equilibrated to room temperature in advance, was added to 96-well white cell culture plates according to the experimental layout at 100. Mu.l/well and incubated at room temperature for 5min in the absence of light.

And (3) performing fluorescence reading by using a multifunctional enzyme-labeled instrument, wherein a chemiluminescent mode is selected for a reading plate mode, an endpoint method is selected for a reading plate type, the wavelength is set to be full wavelength, and fluorescence is collected row by row, and the collection time of each row is 1000ms.

In the experiments described in the above assays, the detection results are shown in fig. 5, where anti-VEGF VHH antibodies LA42F8, LA42F8.5, LA46E11, LA46E11.8 each block VEGF a-induced activation of the KDR signaling pathway.

Example 8 anti-VEGF A VHH inhibition of VEGF A induced HUVEC survival proliferation assay

VEGF A can act on receptors related to VEGFR and the like in vascular endothelial cells, promote the survival, proliferation and migration of the vascular endothelial cells, further induce neovascularization, and the experiment is based on VEGF-induced survival and proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) and detects the inhibition of antibodies on the survival and proliferation of primary cells induced by VEGF A.

The survival and proliferation of HUVECs were determined by CCK-8 in this example as follows: cells HUVEC (Allcells cat# H-001-CN) were treated one day in advance, 2000 cells/well were plated in 96-well plates, incubated in a 5% carbon dioxide incubator at 37℃for 24 hours,

after cell attachment, test media were prepared containing VEGF A at a final concentration of 10ng/ml and antibody (LA 42F8 prepared in example 2, initial concentration 80ug/ml1:3 in equal dilution, negative control IgG, positive control BI836880, group containing VEGF A only at 10ng/ml (VEGFA), and Blank without VEGF A and antibody addition), endothelial cell media in 96-well plates were replaced, incubated in a 5% carbon dioxide incubator at 37℃for 72 hours,

adding CCK-8 detection solution (same kernel chemical number: CK 04) into 10 μl/well, incubating in a 5% carbon dioxide incubator at 37deg.C for 12-24 hr,

Absorbance OD using a multifunctional microplate reader ₄₅₀ -OD ₆₂₀ The value of the read-out value is read out,

in the experiments described in the above assays, the detection results are shown in fig. 6, from which it can be seen that the anti-VEGFA antibody LA42F8 is capable of completely inhibiting VEGFA-induced survival and proliferation of HUVEC cells.

Example 9 anti-VEGF A/Ang2 bispecific antibody HEK293-KDR reporter blocking assay

The chains of the bispecific binding molecules of the invention, IEX04-008, IEX04-010 and IEX04-012 (sequence shown in the sequence listing), were constructed into pcDNA3.1 vectors, which were expressed and purified in 293 cells as described in example 2.

In this example, the blocking effect of anti-VEGF A/Ang2 bispecific antibodies on VEGF A was tested by HEK293-KDR reporter assay. Experimental procedure reference is made to example 7.

The binding molecules or controls used were as follows:

blank: no VEGF a, no antibody;

VEGF A：100ng/ml VEGF A；

negative control IgG: prepared as described above;

positive control Faricimab: prepared as described above, starting at a concentration of 13.5ug/ml,1:3 gradient dilution;

positive control BI-836880: prepared as described above, starting at a concentration of 13.5ug/ml,1:3 gradient dilution;

IEX04-012: prepared as described above, starting at a concentration of 13.5ug/ml,1:3 gradient dilution.

As shown in FIG. 7, the bispecific binding molecule IEX04-012 inhibited VEGF-induced KDR signaling pathway activation, and had better inhibition capacity than control antibody BI 836880.

EXAMPLE 10 anti-VEGF A/Ang2 bispecific binding molecule HUVEC proliferation inhibition assay

The inhibition of VEGF A-induced HUVEC cell survival and proliferation by anti-VEGF A/Ang2 bispecific binding molecules was tested by HEK293-KDR reporter assay.

The experimental procedure was as in example 8, but the antibodies used were as follows:

panel A, IEX04-008, negative control IgG, positive controls Faricimab and BI836880, blank (i.e. no antibody and VEGF A), and VEGF A group (i.e. only 20ng/ml VEGFA added) prepared as described above at an initial concentration of 20ug/ml,1:3 equal dilution;

graph B: initial concentration of 80ug/ml,1:3 equal ratio dilution of negative control IgG, BI-anti-VEGF, IEX04-010, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20ng/ml VEGFA added) prepared as described above;

panel C-IEX 04-012, negative control IgG, positive controls Faricimab and BI836880, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20ng/ml VEGFA added) prepared as described above at an initial concentration of 20nM,1:3 equal dilution.

As shown in FIG. 8, the bispecific binding molecules IEX04-008, IEX04-010, IEX04-012 each inhibited VEGF-induced HUVEC cell survival and proliferation, and IC of IEX04-012 compared to control antibodies BI836880 and Faricimab ₅₀ Lower, have better inhibitory ability.

Example 11 anti-VEGFA/Ang 2 bispecific antibody Ang2 blocking experiments

Ang2 binds to its natural receptor Tie2, and the blocking of binding between Ang2 and Tie2 by anti-VEGF a/Ang2 bispecific binding molecules was examined by ELISA and FACS.

(1)ELISA

The ability of IEX04-008, IEX04-010 and IEX04-012, and control antibodies BI-836880 and Faricimab to block binding of human Ang2 to hTie2 was tested by ELISA.

The hTie2 protein (beijing-sense-forsythia) was resuspended and dissolved to a concentration of 2ug/ml using PBS and coated onto the elisa plate overnight. The biotin antigen Recombinant Biotinylated hAngiopoietin-2 protein (R & D) was diluted to 600ug/ml M,50 ul/well using 5% BSA blocking for 1 h. Antibodies prepared as described above (IEX 04-008, IEX04-010, IEX04-012 and positive control antibodies BI836880 and Faricimab and negative control IgG) were prepared from the highest concentration of 300nM1:2, total 8 or 12 dilution gradients, 50 μl/well, incubation on PBS ice for 30min, final biotin antigen concentration 300ng/ml. The antigen-antibody mixture obtained as described above was incubated to an ELISA plate for 90min, washed three times with PBS, the supernatant was discarded, 100. Mu.l Avidin-HRP (Invitrogen)/well diluted 1:10000 was added, and washed six times with PBS at room temperature for 30 min. Color development was performed for 1min using 100 ul/well TMB color development solution (Solarbio) and 100 ul/Kong Zhongzhi using stop solution (Solarbio).

Reading OD for each well using a microplate reader ₄₅₀ ，OD ₆₂₀ 。

The experimental results show (see FIG. 9) that IEX04-008, IEX04-010, IEX04-012 and control antibody BI836880 all have complete blocking effect, and that the ICs of the bispecific binding molecules of the invention ₅₀ Are significantly smaller than the positive control antibody.

(2) Flow cytometry detection (FACS)

The ability of IEX04-008, IEX04-010, IEX04-012 and positive control antibodies BI-836880 and Faricimab and negative control IgG to block binding of human Ang2-hFc to Tie2 on the cell surface was examined by FACS.

The antigen hAN 2-Fc protein (Beijing Yiqiao, cat# 10691-H02H) was diluted to 4ug/ml,50 μl/well. Antibodies prepared as described above (IEX 04-008, IEX04-010, IEX04-012, positive control antibodies BI-836880 and Faric)imab and negative control IgG) were 2-fold gradient diluted from a maximum concentration of 800nM, a total of 12 dilution gradients, 50 μl/well, incubated on PBS ice for 30min, antigen-hAng 2-Fc protein final concentration of 2ug/ml, and a maximum final concentration of 400nM per antibody. The 293-Tie2 cells prepared as described above were conditioned to 2X 10 ⁵ Cells/well, 100 μl/well. Cells were centrifuged at 300g for 5min, the supernatant was discarded, and resuspended in antigen-antibody mixture. Incubation on ice for 30min, adding 100. Mu.l/well PBS, centrifuging for 5min at 300g, washing with PBS for 1 time, adding 100. Mu.l of Goat anti-human IgG-PE (SouthernBiotech)/well diluted 1:200, ice-bathing for 20min, adding 100. Mu.l/well PBS, centrifuging for 5min at 300g, washing with PBS for 1 time. Cell fluorescence signal values were detected by a cell flow cytometer (BD Biosciences) resuspended in 100. Mu.l PBS. Concentration-dependent curves were fitted with GraphPad according to their MFI. The results are shown in FIG. 10. The bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 are shown to be effective in blocking binding of human Ang2-hFc to Tie2, and IC ₅₀ Lower than the positive control.

Example 12 anti-VEGF A/Ang2 bispecific binding molecule Ang2 phosphorylation inhibition assay

This example demonstrates the inhibition of the two-specific binding molecules of the invention on the hAng2-Fc induced Tie2 phosphorylation, which employs a hAng 2-induced phosphorylation assay.

The double-specificity binding molecules and the recombinant hAN 2-Fc protein are incubated together to overexpress Tie2 from the Expi293 cells, 293-Tie2, and the content of phosphorylated Tie2 in the system is detected, so that the inhibition effect of different antibodies on the hAN 2-Fc induced Tie2 phosphorylation is reflected.

The overexpressed 293-Tie2 cells prepared as described above were diluted to 2X 10 ⁶ cell/ml, 100ul of 96-well plates were added per well, centrifuged at 400g for 5min, and the supernatant removed.

The experimental medium was prepared using an Expi293 medium (Thermo cat No. a 1435102) to which the test antibodies (IEX 04-012, positive control BI-836880 and Faricimab, negative control IgG prepared as above) were added at a maximum final concentration of 60ug/ml, followed by 1:2 equal dilution, and had 2-Fc (beijing je cat No. 10691-H02H) at a final concentration of 2.5ug/ml.

Cells were resuspended in 100ul of experimental medium per well, incubated at 37℃for 15min, centrifuged to remove medium, 100ul of NP-40 lysate containing 1% protease and phosphatase inhibitors was added and placed on ice for 30min. Centrifuging at 2000g, collecting protein supernatant, storing in-80 deg.C refrigerator,

The concentration of pTie2 was detected according to the instructions of the phosphorylated Tie2 ELISA kit (R & D DYC 2720E) and the capture antibody was coated onto the ELISA plate at a concentration of 4ug/ml for 4℃overnight. PBST was washed three times and blocked with 5% bsa for 1h. 100ul of sample to be tested and control pTie2 (R & D DYC 2720E) were added and used to make a standard curve and incubated for 2h at room temperature (if the concentration of sample pTie2 is too high, and exceeds the ELISA detection range, the obtained mixture may be diluted 2-3 times). PBST was washed three times, 100ul of HRP-conjugated anti-pTyr antibody (R & D cat# DYC 2720E) was added and incubated for 2h at room temperature. PBST was washed 6 times, developed by adding 100ul TMB, and after 15min, the reaction was stopped by adding 100ul stop buffer. The OD450-OD620 per well was measured using a spectrophotometer.

The experimental results are shown in FIG. 11. The antibody IEX04-012 of the invention can effectively inhibit 293-Tie2 phosphorylation induced by hAN 2-Fc in vitro, and IC ₅₀ Is superior to positive control.

Example 13 inhibition of vascular endothelial cell leakage by anti-VEGF A/Ang2 bispecific binding molecules

The present study identified the effect and function of anti-VEGF A/Ang2 bispecific binding molecules on vascular endothelial cell leakage by HUVEC-Tie2 leakage experiments.

HUVEC cells (Allcels cat# H-001-CN) were transfected with lentivirus to obtain HUVEC-Tie2 cells overexpressing Tie 2.

The HUVEC-Tie2 was digested with Accutase (Sigma) and obtained using 300ul of EGM-2 medium spread under mini-well 96-well insert dishes and resuspended to 1X 10 using EGM-2 medium ⁷ Cells/ml, 100 ul/well were plated on top of the dish. The lower chamber medium (EGM-2 medium) was replaced every 24 hours, and after 24 hours the lower chamber medium was replaced with experimental medium consisting of:

blank: EGM-2 medium (Lonza cat# CC-5035),

VEGF a group: EGM-2 Medium+20 ng/ml VEGF (R & D cat# 293-VE)

IgG group (VEGF a+igg): EGM-2 medium+20 ng/ml VEGF+10ug/ml IgG;

ang1 group (VEGF a+ang 1): EGM-2 medium+20 ng/ml VEGF (R & D cat# 293-VE) +200ng/ml Ang1 (R & D cat# 923-AN);

IEX04-012 group (VEGF a+iex04-012): EGM-2 Medium+20 ng/ml VEGF+10ug/ml IEX04-012;

BI-836880 group (VEGF A+BI-836880): EGM-2 medium+20 ng/ml VEGF+10ug/ml BI-836880;

set of Faricimab (VEGF a+faricimab): EGM-2 Medium+20 ng/ml VEGF+10ug/ml Faricimab.

The experimental culture medium is placed at 37 ℃ and 5% CO ₂ Culturing.

After 24 hours 1ul of FITC-Dextran (Sigma cat# FD 2000S-1G) (4 mg/ml) was added to each well of the upper laboratory medium and the mixture was placed at 37℃in 5% CO ₂ Taking down the room culture medium after 30min, diluting with PBS (phosphate buffer solution) for 1:10, and detecting with a multifunctional enzyme-labeled instrument, wherein the excitation wavelength is 488nm, and the emission wavelength is 535nm.

The experimental results are shown in FIG. 12, which shows that the antibody IEX04-012 of the present invention is effective in reducing VEGF-induced vascular endothelial cell permeability.

EXAMPLE 14 laser-induced choroidal neovascularization efficacy test

The experiment uses a rhesus laser-induced choroidal neovascularization model to measure the anti-neovascularization effect of the bispecific binding molecule IEX04-012 of the present invention.

Rhesus monkey:

species: rhesus monkey; grade: a normal stage; weight of: the weight of the bicycle is 3.30-4.20 kg when the bicycle is purchased; the weight of the mold is 3.35-4.35 kg during molding; the source is as follows: license number is produced by Sichuan horizontal and vertical biotechnology shares limited company: SCXK (chuanli) 2019-029, experimental animal quality eligibility number: no.0023356;

the test adopts laser to surround the central fovea of the fundus macula of rhesus to induce the ocular fundus choroid to be angiogenesis, and establishes an animal model similar to the human choroid neovascularization. Fluorescein fundus angiography is carried out before and 20 days after photocoagulation to judge the molding condition, and 20 rhesus monkeys (male and female halves) with successful molding are selected to be divided into 5 groups, namely a model control group, an IEX04-012 low dose group, an IEX04-012 high dose group, an Elyea group and a Faricimab group, wherein 4 monkeys are respectively in each group, and the male and female halves.

On day 21 after photocoagulation, the monkeys of each group were dosed as indicated in the table, and IEX04-012, eylea (Bayer) or Faricimab (both dissolved in 0.9% sodium chloride injection) were given by vitreous injection to both eyes, and the model control group was given an equal volume of 0.9% sodium chloride injection. The animals of each group were subjected to fundus color photography, fluorescein fundus angiography (plaque statistics and leak area measurement) 7, 14, 21, 28 days after administration (Robin J good, wenzheng Hu, afshin Shadie et al Optimization of laser-ind uced choroidal neovascularization in African green monkies. Experimental Eye Research, exp Eye Res.2011 92 (6): 464-72), optical coherence tomography (OCT, wang Q, lin X, xiang W et al Assessment of laser induction of Bruch's membrane disruption in monkey by spectral-domain optical coherence tomogram. British Journal of Ophthal mology,2015,99 (1): 119-24), and the test subjects were observed for inhibition of choroidal neovascularization. Histology with immunohistochemical (HE) staining was performed on both eyes after euthanasia at 29 days post-dose.

Experimental design table

As shown in fig. 13-15, bispecific binding molecules of the invention showed significant anti-neo-vascularization immediately after 28 days of administration, and statistics of the number of four grade leakage spots (fig. 13A) and three to four grade leakage spots (fig. 13B) showed that the IEX04-012 group treated animals had significantly less high leakage spots than the control and positive control administration groups. OCT results showed that IEX04-012 treated animals showed a significant decrease in retinal thickness suggesting a reduction in retinal edema and an effect superior to the control (fig. 14). The result of fluorescein fundus angiography shows that the fundus leakage area of the IEX04-012 treatment group is obviously reduced, and the effect is superior to that of positive controls Eylea and Faricimab (figure 15), which shows that the antibody of the invention can obviously inhibit leakage caused by new blood vessels. In conclusion, the antibody provided by the invention has obvious inhibition effect on fundus neovascularization induced by laser by combining the antibody with the anti-VEGF inhibitor, and has the function of protecting vascular integrity.

The rhesus monkeys were anesthetized with pentobarbital sodium (about 30mg/kg by intravenous injection, the dose of which was adjusted according to the health condition of the animals) 29 days after administration, and the abdominal aorta or femoral artery was euthanized for general observation, and bilateral eyeballs were extracted.

Part of the animals were fixed with modified Davidson's fixative, paraffin embedded sections, and laser molded areas were selected for routine HE staining, CD31 IHC staining, and other histopathological examination.

The antibody group of the invention showed better morphological improvement of retina compared with anti-VEGF single treatment in pathological section, obviously reduced retina pathologic area, reduced retina edema and reduced tissue proliferation in laser injury area (see figure 16), inhibited retinal choroidal neovascularization and enhanced vascular integrity function (figure 17).

Sequence listing

Claims

A VHH antibody against Ang2 comprising 3 CDRs, HCDR1, HCDR2 and HCDR3, wherein

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 16;

HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 17 or 20;

HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 18.
The VHH antibody of claim 1, wherein the VHH

(1) Comprising the amino acid sequence set forth in SEQ ID NO. 19 or 21, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 19 or 21, or consisting of the amino acids set forth in SEQ ID NO. 19 or 21; or (b)

(2) Comprising an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO. 19 or 21.
A bispecific binding molecule binding to VEGFA and Ang2 comprising a first target binding domain that specifically binds VEGF A and a second target binding domain that specifically binds Ang2, wherein the second target binding domain is a VHH antibody of claim 1 or 2,

optionally, wherein the first target binding region is selected from the group consisting of:

VHH that specifically binds VEGFA;

an antigen-binding fragment of an antibody that specifically binds VEGFA, e.g., an scFv, e.g., the antibody is a fully human antibody or a humanized antibody; or (b)

VEGF receptor (VEGF R) or an extracellular domain thereof or a fusion protein comprising an extracellular domain thereof, e.g. a fusion protein of an extracellular domain thereof with Fc, which specifically binds to VEGF a.
The bispecific binding molecule of claim 3 which is a bispecific antibody.
The bispecific binding molecule of claim 3 or 4 which is bivalent, trivalent or tetravalent.
The bispecific binding molecule of claim 4 or 5 having the structure:

light chain variable region of anti-VEGF antibody VL-linker-heavy chain variable region of anti-VEGF antibody VH-linker-anti-Ang 2 VHH or

Heavy chain variable region of anti-VEGF antibody VH-linker-anti-VEGF antibody light chain variable region VL-linker-anti-Ang 2 VHH.
The bispecific binding molecule of claim 6, wherein:

the light chain variable region VL of an anti-VEGF antibody comprises or consists of LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the sequence set forth in SEQ ID NO. 31; LCDR2 comprises or consists of the sequence shown in SEQ ID NO. 32; LCDR3 comprises or consists of the sequence shown in SEQ ID NO. 33; and/or

The heavy chain variable region VH of the anti-VEGF antibody comprises or consists of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID No. 35; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 36; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 37.
The bispecific binding molecule of claim 6 or 7, wherein

The heavy chain variable region VH of the anti-VEGF antibody comprises or consists of an amino acid sequence set forth in SEQ ID No. 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID No. 34; or said heavy chain variable region VH comprises an amino acid sequence having one or several (preferably NO more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID No. 34, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions;

The light chain variable region VL of an anti-VEGF antibody comprises or consists of an amino acid sequence set forth in SEQ ID NO. 30, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 30; or the light chain variable region VL comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID No. 30, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
The bispecific binding molecule of any one of claims 6 to 8, wherein

(1) The binding molecule comprises the amino acid sequence of SEQ ID NO. 28, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 28, or consists of the amino acids of SEQ ID NO. 28; or (b)

(2) The bispecific binding molecule comprises an amino acid sequence having one or several, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations compared to the amino acid sequence shown in SEQ ID NO. 28, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
The bispecific binding molecule of claim 4 or 5 having the structure:

a first anti-VEGF VHH-linker-second anti-VEGF VHH-linker-anti-Ang 2 VHH,

wherein the first anti-VEGF VHH is the same as or different from the second anti-VEGF VHH.
The bispecific binding molecule of claim 10, wherein the first anti-VEGF VHH or the second anti-VEGF VHH comprises HCDR1, HCDR2 and HCDR3, wherein

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 1; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 2; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 3;

or alternatively

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 6; HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 7 or 10; HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 8.
The bispecific binding molecule of claim 10 or 11, wherein the first anti-VEGF VHH or the second anti-VEGF VHH comprises or consists of an amino acid sequence of SEQ ID No. 4, 5, 9 or 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID No. 4, 5, 9 or 11; or the VHH comprises an amino acid sequence having one or several (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence shown in SEQ ID No. 4, 5, 9 or 11.
The bispecific binding molecule of any one of claims 10 to 12, wherein

(1) The binding molecule comprises the amino acid sequence of SEQ ID NO. 22, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 22, or consists of the amino acids of SEQ ID NO. 22; or (b)

(2) The bispecific binding molecule comprises an amino acid sequence having one or several, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations compared to the amino acid sequence shown in SEQ ID NO. 22, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
The bispecific binding molecule of claim 3 comprising one or two of the following chains:

VEGF R extracellular domain-Fc-linker-anti-Ang 2 VHH.
The bispecific binding molecule of claim 14, wherein the VEGFR extracellular domain is an extracellular domain of a human-derived VEGFR; preferably, the VEGFR extracellular domain comprises a VEGFR1 second antibody-like domain and a VEGFR2 third antibody-like domain; more preferably, the VEGFR extracellular domain comprises a human VEGFR1 secondary antibody-like domain and a human VEGFR2 tertiary antibody-like domain.
The bispecific binding molecule of claim 14 or 15, wherein the VEGFR extracellular domain comprises the amino acid sequence of SEQ ID No. 26 or comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 26.
The bispecific binding molecule of any one of claims 14 to 16, wherein the Fc is an Fc of human IgG1, igG2, igG3 or IgG4 origin, preferably the Fc comprises the amino acid sequence of SEQ ID No. 27, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 27, or consists of the amino acids of SEQ ID No. 27.
Bispecific binding molecule according to any one of claims 14 to 17, wherein the VEGF R ectodomain-Fc is a fusion protein of a VEGFR ectodomain with Fc, such as for example Aflibercept or a derivative thereof, such as comprising the amino acid sequence of SEQ ID No. 25, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of said SEQ ID No. 25, or consisting of the amino acids of said SEQ ID No. 25.
The bispecific binding molecule of any one of claims 14 to 18, wherein

(1) The binding molecule comprises the amino acid sequence of SEQ ID NO. 24, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 24, or consists of the amino acids of SEQ ID NO. 24; or (b)

(2) The bispecific binding molecule comprises an amino acid sequence having one or several, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations compared to the amino acid sequence shown in SEQ ID NO. 24, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
The bispecific binding molecule of any one of claims 3 to 19, wherein the linker comprises or consists of the amino acid sequence of SEQ ID No. 23.
A VHH antibody against VEGF a comprising 3 CDRs, HCDR1, HCDR2 and HCDR3, wherein

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 1;

HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 2;

HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 3;

Or alternatively

HCDR1 comprises or consists of the sequence shown in SEQ ID NO. 6;

HCDR2 comprises or consists of the sequence shown in SEQ ID NO. 7 or 10;

HCDR3 comprises or consists of the sequence shown in SEQ ID NO. 8.
The VHH antibody of claim 21, which

(1) Comprising the amino acid sequence of SEQ ID NO. 4, 5, 9 or 11, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 4, 5, 9 or 11, or consisting of the amino acids of SEQ ID NO. 4, 5, 9 or 11; or (b)

(2) Comprising an amino acid sequence having one or several, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mutations compared to the amino acid sequence shown in SEQ ID NO. 4, 5, 9 or 11, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
A nucleic acid molecule encoding a VHH antibody according to any one of claims 1 or 2 or 21 or 22, or a bispecific binding molecule according to any one of claims 3 to 20 or a chain of said binding molecules.
An expression vector comprising the nucleic acid molecule of claim 23, preferably said expression vector is pcdna3.1.
A host cell comprising the nucleic acid molecule of claim 23 or the expression vector of claim 24, preferably the host cell is prokaryotic or eukaryotic, e.g. a bacterium such as an e.coli cell, e.g. TG1; or 293 cells, such as 293F or Expi-293 cells.
A method of making a VHH antibody according to any one of claims 1 or 2 or 21 or 22, or a bispecific binding molecule according to any one of claims 3 to 20, the method comprising culturing a host cell according to claim 25 under conditions suitable for expression of the VHH antibody or bispecific binding molecule or a chain thereof, and optionally recovering the VHH or bispecific binding molecule from the host cell (or host cell culture medium).
An immunoconjugate comprising the VHH antibody according to any one of claims 1 or 2 or 21 or 22, or the bispecific binding molecule according to any one of claims 3 to 20
A pharmaceutical composition or formulation comprising a VHH antibody according to any one of claims 1 or 2 or 21 or 22, or a bispecific binding molecule according to any one of claims 3 to 20, and optionally one or more other therapeutic agents, and optionally a pharmaceutical excipient.
A method of preventing or treating an ocular disease in a subject comprising administering to the subject an effective amount of a VHH antibody according to any one of claims 1 or 2 or 21 or 22, or a bispecific binding molecule according to any one of claims 3 to 20, an immunoconjugate according to claim 27 or a pharmaceutical composition or formulation according to claim 28.
The method of claim 29, wherein the ocular disease is selected from ocular diseases associated with angiogenesis, such as ocular diseases associated with corneal neovascularization.
The method of claim 29 or 30, wherein the patient has (e.g., elevated levels of, e.g., nucleic acid or protein levels of) VEGF, e.g., VEGF a, and/or Ang2.
The method of any one of claims 29-31, wherein the method further comprises administering one or more therapies, such as therapeutic regimens and/or other therapeutic agents, to the patient.